Determination of pili by conjugative bacterial drug resistance plasmids of incompatibility groups B, C, H, J, K, M, V, and X.

Representative plasmids from incompability groups B, C, H, J, K, M, V, and X were transferred to "bald" strains of Escherichia coli or Salmonella typhimurium. By using a new technique, pili were detected by electron microscopy for each incompatibility group. Morphology varied but was similar for plasmids within a group. These findings suggest that all conjugative plasmids in the Enterobacteriaceae may determine pili.     

Naturally occurring plasmids exhibiting incompatibility with members of incompatibility groups I and P

From a group of naturally occurring antibiotic resistance plasmids, a number of plasmids were identified which were incompatible with members of incompatibility group P and also incompatibility group I alpha or I gamma. These plasmids also exhibited strong entry exclusion with members of group P only and showed a host range which resembled that of plasmids of group I rather than those of group P. Segregants of a number of these plasmids appeared to have lost some of the incompatability and/or surface exclusion functions. Studies of nucleic acid homology indicated that these plasmids were very similar to one another. They exhibited 15 to 20% nucleic acid homology with representatives of the I alpha and I gamma groups, yet showed less than 2% homology with the group P plasmid RP4.     

Recombination between plasmids of incompatibility groups P-1 and P-2.

R plasmids of incompatibility group P-2 are readily transmissible between Pseudomonas strains, but not to Escherichia coli or other enterobacteria, whereas those of group P-1 have a broad host range. Pseudomonas aeruginosa donor strains carrying both a P-1 plasmid (RP1, RP4, or R751) and a P-2 plasmid (pMG1, pMG2, pMG5, or RPL11) were mated with E. coli K-12, and selection was imposed for resistance markers on the P-2 plasmids. Transconjugants were obtained at a low frequency, in which P-2 markers were expressed and were serially transmissible in E. coli together with P-1 markers. These plasmids had P-1 incompatibility properties, conferred susceptibility to phages active on P-1 carrying strains, and behaved on sucrose gradient centrifugation as unimolecular species of higher molecular weights than the P-1 parent. Recombinant plasmid formation was independent of a functional Rec gene in both donor and recipient and, with R751, had a preferred site leading to loss of trimethoprim resistance. Interaction between insertion sequences may be involved. Thus, plasmids of group P-2 can recombine with R factors of another group quite separate in compatibility properties, host range, and pilus type. Formation of such recombinants provides one pathway by which the genetic diversity of plasmids may have evolved.     

Evidence for two genetically distinct DNA primase activities specified by plasmids of the B and I incompatibility groups.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.     

Reconsideration of the previously classified incompatibility groups of plasmids,IncP-1 and IncP-11

This study presents the reassessment of earlier published data with reference to the article published in Environmental Microbiology entitled ‘IncP-type plasmids carrying genes for antibiotic resistance or aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains’ by Lo et al. This correspondence clarifies misperceptions of plasmids classified under incompatibility (Inc) groups IncP-1 and IncP-11.     

Demonstration of a third incompatibility function on plasmids already incompatible with group P and group I plasmids

The addition of extra antibiotic resistance determinants to plasmids of a series previously shown to be incompatible in an atypical fashion with group I and group P plasmids, has enabled the existence of a third and major incompatibility function determined by these plasmids to be demonstrated. The in vitro construction of a plasmid consisting of the replication region of one of the plasmids, linked to the genes of the galactose operon, facilitated the identification of this third incompatibility function as similar to IncB plasmids.     

Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5.

Large plasmids from Agrobacterium tumefaciens, Salmonella typhimurium, Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa were routinely and consistently isolated using a procedure which does not require ultracentrifugation but includes steps designed to separate large-plasmid DNA from the bacterial folded chromosome. It also selectively removes fragments of broken chromosome. A variety of large plasmids was readily visualized with agarose gel electorphoresis, including five between 70 and 85 megadaltons (Mdal) in size, six between 90 and 143 Mdal, one that was larger than 200 Mdal, and one that was larger than 300 Mdal. This isolation procedure allowed initial estimation of the molecular sizes of the two IncP2 plasmids, pMG1 and pMG5, which were 312 and 280 Mdal, respectively. A standard curve for size determination by gel electrophoresis including plasmids between 23 and 143 Mdal in size did not extrapolate linearly for plasmids of the 300-Mdal size range. Unique response of different plasmids to the isolation procedure included sensitivity of IncP1 plasmids to high pH and the co-isolation of a 20-Mdal "cryptic" plasmid in conjunction.     

Serological characteristics of pili determined by the plasmids R711b and F0lac.

The plasmids R711b (at present IncX) and F0lac (IncFV) both determine pili morphologically like those of F (IncFI), and confer sensitivity to the F-specific filamentous bacteriophages, but not to the F-specific isometric RNA phages. Detailed serological studies show that the two pilus types are unrelated, and that neither is related to any of the previously defined F pilus serotypes. Adsorption of the isometric RNA phage MS2 to R711b pili occurs in the presence but not in the absence of formalin, which presumably prevents elution of reversibly adsorbed virions. No adsorption occurs with F0lac pili. MS2 multiplication, as measured by titre increase tests in liquid medium, is found with neither plasmid. The two plasmids are not incompatible. These observations indicate that R744b and F0lac are different both from one another and from the plasmids belonging to the incompatibility groups IncFI--IV.     

Characterization of pili determined by drug resistance plasmids R711b and R778b.

The bacterial drug resistance plasmids R711b and R778b, at present classified in the X incompatibility group, determine pili (designated 711) that resemble F pili morphologically. Like F pili, 711 pili adsorb F-specific filamentous bacteriophages to their tips, though more often in pairs, than singly. However, F-specific RNA-containing bacteriophages are not adsorbed to their sides, and strains carrying the plasmids are resistant to these phages. Pili determined by the only IncFV plasmid Folac are similar to 711 pili in their phage adsorption properties, but they are serologically different, as are F pili. It is concluded that F, Folac and 711 pili have basic differences in spite of a morphological resemblance.     

Morphology of pili determined by the N incompatibility group plasmid N3 and interaction with bacteriophages PR4 and IKe

The IncN plasmid N3 was transferred to bald strains of Salmonella typhimurium LT2 and Escherichia coli K-12. In both cases, transconjugants were found to carry short pili, which were designated N pili. They were very easily detached from cells and readily broken into short pieces. N pili were found to be straight inflexible rods 9.5 nm thick and sharply pointed at the distal end. The N-specific filamentous bacteriophage IKe and the lipid-containing phage PR4 both adsorbed to the pointed tips when mixed with cell-free suspensions of N pili.     

Suppression and enhancement of temperature sensitivity of dnaB mutations of Escherichia coli K12 by conjugative plasmids.

A majority of wild-type, conjugative antibiotic-resistance plasmids from a standard collection had the ability to suppress or to enhance the temperature sensitivity of dnaB mutants of Escherichia coli K12. This ability appears to be widely dispersed among all plasmid groups. The mode of suppression does not involve the insertion of the plasmid into the chromosome. Plasmid-induced suppression or enhancement is not as a rule mutation specific and can extend to several mutations within the dnaB region. The patterns of suppression and enhancement suggest a direct or indirect interaction between a plasmid-specified product and the dnaB protein.     

Enhancement of UV survival, UV- and MMS-mutability, precise excision of Tn10 and complementation of umuC by plasmids of different incompatibility groups

29 conjugative resistance and colicin plasmids from 19 different incompatibility (Inc) groups were examined for their ability to enhance post-ultraviolet (UV) survival and UV- and methyl methanesulfonate(MMS)-induced mutability in Salmonella typhimurium LT2 strains. 14 Muc+ plasmids enhanced each of the survival and mutation-related properties tested, while 14 Muc- plasmids showed no enhancing effects in any tests. One Muc+ plasmid, pRG1251 (IncH1), enhanced post-UV survival and each of the mutation-related properties tested, except MMS-induced mutagenesis. Two further noteworthy plasmids, R391 (IncJ) and R394 (IncT), produced apparent strain-dependent effects in S. typhimurium which differed from those reported to have been found in Escherichia coli. Plasmid R391 enhanced post-UV survival in S. typhimurium, in contrast to its UV-sensitizing effects in E. coli. In both hosts plasmid R391 enhanced UV- and MMS-induced mutagenesis. Plasmid R394 had no enhancing effects on UV survival or UV- and MMS-induced mutagenesis in S. typhimurium, in contrast to its reported enhancement of MMS-induced mutagenesis in E. coli. Conjugal transfer of R394 to E. coli strain AB1157 and assays of mutagenesis-related traits supported results observed in S. typhimurium. Muc+ plasmids were found to enhance the frequency of precise excision of the transposon Tn10 when inserted within hisG or trpA in S. typhimurium strains. Precise excision could be further enhanced in S. typhimurium by UV-irradiation. Analysis of Tn10 mutants with altered IS10 ends indicated that intact inverted repeats at the ends of Tn10 were required for efficient enhancement of precise excision.     

Evidence that the hemolysin/bacteriocin phenotype of Enterococcus faecalis subsp. zymogenes can be determined by plasmids in different incompatibility groups as well as by the chromosome.

The hemolysin (Hly/Bac) determinant in strains of Enterococcus faecalis was found to be present on plasmids in different incompatibility groups (conferring different sex pheromone responses) as well as on the chromosome. Of 33 Hly/Bac plasmids identified in clinical isolates, the related pheromone for 30 was cAD1; the related pheromone for another two (pYI1 and pYI3) or one (pYI2) was cOB1 or cY12, respectively. The representative Hly/Bac plasmids pAD1, pYI1, pOB1, and pYI2, which responded to pheromones cAD1, cOB1, cOB1, and cYI2, respectively, were compatible with one another. As additions to the incompatibility group IncHly of pAD1, groups for pOB1, pYI1, and pYI2 were designated IncHlyII, IncHlyIII, and IncHlyIV, respectively. Eleven of the 30 plasmids conferring a response to cAD1 were very similar to pAD1 on the basis of their restriction endonuclease profiles. EcoRI fragment D, F, or H containing parts of the Hly/Bac gene(s) of pAD1 hybridized to similar EcoRI fragments from each of the other three representatives of incompatibility groups (i.e., pOB1, pYI1, and pYI2) and to homologous DNA representing the chromosome of the plasmid-free Hly/Bac strain YI6-1.     

Restriction endonuclease analyses of the incompatibility group P-1 plasmids RK2, RP1, RP4, R68, and R68.45

The P-group plasmids RP1, RP4, RK2, R68 and R68.45 were analyzed by the following restriction endonucleases:BamHI,BglII,EcoRI,HindIII,PstI,PvuII,SalI, andSmaI. No differences between RP1, RP4, and RK2 were found, and the plasmid R68.45 was found to contain a direct duplication of an existing DNA sequence in R68. Our map of RK2 differs from the published map of RK2 in the corresponding region of the R68 map that is duplicated in R68.45.     

Generation of the HLA-B35, -B5, -B16, and B15 groups of alleles studied by intron 1 and 2 sequence analysis

 HLA-B is the most polymorphic of the major histocompatibility complex classical class I loci. This polymorphism is mainly in exons 2 and 3, which code for the molecule’s α1 and α2 domains and include the antigenic peptide binding site. Recent studies have indicated that not only exons but also the intron 2 region may be involved in the generation of certain HLA-B alleles such as B ^* 3906 and B ^* 1522. To study the degree of intron 2 participation and the mechanisms that generate polymorphism at the HLA-B locus, intron 1 and 2 sequences from the HLA-B35, -B5, -B16 and -B15 groups of alleles were obtained. A group-specific intronic polymorphism was found: namely, B ^* 5301 shows intron 1 and 2 sequences identical to those found in all B35 alleles studied. On the other hand, B ^* 5101 and B ^* 52012 show the same intron 1 and 2 sequences and their intron 1 is the same as that found in the B35 group. This suggests that B5 and B35 groups of alleles may have arisen from a common ancestor. All known B16 alleles show the same introns 1 and 2, with the exception of B ^* 39061 and B ^* 39062, and all B15 alleles also bear the same introns 1 and 2, with the exception of B ^* 1522. Variability at intron 1 is more restricted than at intron 2, and the use of intron 1 for HLA-B allele phylogenetic analysis is better for grouping alleles of a postulated common origin. In conclusion, there is a remarkable conservation of intronic sequences within related HLA-B alleles, which probably reflects a common origin and perhaps a selective force avoiding DNA changes. Intronic sequences are also potentially useful to design DNA typing strategies. Received: 11 March 1997 / Revised: 29 May 1997     

Cytochalasins Z1, Z2 and Z3, three 24-oxa[14]cytochalasans produced by Pyrenophora semeniperda

Three new cytochalasans, named cytochalasins Z1, Z2 and Z3, were isolated from the wheat culture of Pyrenophora semeniperda, a fungus proposed to biologically control grass weeds. Other cytochalasins isolated from the same organic extract were identified as the already known cytochalasins F, T, deoxaphomin and cytochalasins B, the latter being produced in very large amounts. All three new cytochalasins were characterized as 24-oxa[14]cytochalasans by extensive use of NMR and MS techniques. Cytochalasins Z1 and Z2 proved to be structurally related to cytochalasin T, whereas cytochalasin Z3 was related to cytochalasin B. When assayed on wheat and tomato seedlings, cytochalasin Z3, in comparison to the new cytochalasins, cytochalasin B, its 21,22-dihydroderivative, cytochalasin F and deoxaphomin showed a remarkable ability to inhibit root elongation. The possibility of using these metabolites in biological control strategies is discussed.